Heat Shock Proteins Heat shock proteins (hsp) are ubiquitous polypeptides produced by all cells of all species. They are among the best-conserved proteins phylogenetically, with respect to both sequence and function. Human and bacterial Heat shock proteins are more than 50% homologous (Jindal et AL. Mol Cell Biol. 9: 2279, 1989). Heat shock proteins are expressed both as constitutive proteins that act as molecular chaperones (Becker J. and Craig A. E., Eur. J. Biochem. 219; 11, 1994), and as inducible stress proteins.
Hsp60 is a mitochondrial chaperone with a major role in protein folding and unfolding as well as translocation of proteins into mitochondria. Hsp60 is found in the cell cytosol under stressful and inflammatory conditions; infection or elevated cytokine levels will induce the cellular stress response. Therefore, it is not surprising that hsp60 is a highly immunogenic protein: it is the “common antigen” of gram-negative bacteria.
Immunological reactivity to both bacterial and autologous-hsp60 is highly prevalent in the general population, since the pathogen-directed immune response can easily convert into an autoimmune response due to the high homology.
T-cell responses to multiple hsp60 epitopes are present in various autoimmune and inflammatory diseases (Van Eden et al. Immunology Today 19; 303, 1998), including type 1 diabetes (Elias et al. Proc. Natl. Acad. Sci. 88: 3088, 1991), rheumatoid and juvenile arthritis, multiple sclerosis, ankylosing spondylitis, pelvic inflammation-associated infertility, inflammatory bowel disease, atherosclerosis, graft rejection and more. The immune system reacts to hsp60 epitopes that are either cross-reactive between the human and bacterial analogues, or idiosyncratic.
Inflammatory diseases associated with hsp60 expression in target tissues include: (i) Autoimmune diseases: diabetes (Birk et al. Proc Natl Acad Sci 93: 1032, 1996), multiple sclerosis, rheumatoid arthritis (Van Eden et al., Nature, 331: 171, 1988), juvenile chronic arthritis; (ii) Chronic inflammation: inflammatory bowel disease, reactive arthritis; (iii) Graft rejection: Hsp60 can contribute significantly to the inflammatory process that ends in graft rejection. By introducing hsp60-derived antagonists at an early stage after transplantation, one could dampen the inflammation and prolong graft survival (Birk et al. Proc Natl Acad Sci, 96: 5159, 1999); (iv) Atherosclerosis: Hsp60 has been implicated in atherosclerosis, since autoantibodies to human hsp60 were demonstrated to correlate with the clinical status of patients and experimental animal models. Moreover, hsp60 can stimulate macrophage functions relevant to atherosclerosis, such as the production of TNFα, IL-6 and matrix-degrading metalloproteinases (Chen et al., J. Immunol. 162, 3212, 1999).
Use of Heat Shock Proteins in Therapy
Many disclosures claim uses of heat shock proteins or fragments thereof as immune modulators in diagnosis, treatment or prevention of autoimmune diseases. Most of these disclosures relate to heat shock protein 60 also known previously as hsp65, or fragments of this protein.
For example, the particular protein produced by the human body during development of IDDM, which serves as a diagnostic marker for the incipient outbreak of IDDM, is the human heat shock protein having a size of about 65 KD (human hsp65) or an antigen cross-reactive therewith as disclosed in EP 0417271, and in U.S. Pat. Nos. 5,114,844; 5,671,848; 5,578,303 and 5,780,034. It has been disclosed that fragments of this hsp60 protein may serve as therapeutically useful entities in preventing or alleviating IDDM and host vs. graft disease (U.S. Pat. Nos. 6,180,103 and 5,993,803 and WO 96/19236, WO 97/01959 and WO 98/08536).
In addition, fragments of hsp60 may be used as carriers for development of synthetic vaccines by increasing the immunogenicity of poorly immunogenic antigens as disclosed in U.S. Pat. Nos. 5,736,146 and 5,869,058.
European Patent No. 0262710 discloses polypeptides useful for alleviation, treatment, and diagnosis of autoimmune arthritis and similar autoimmune diseases. The claimed polypeptides are derived from bacterial protein named “Antigen A” which was identified later as mycobacterial hsp60.
WO 92/04049 discloses peptides of at least seven amino acids homologous to a fragment of Mycobacterium tuberculosis hsp60, which inhibit T-lymphocytes activation and proliferation and can protect from immune reactions and immune-related disease.
WO 89/12455 and WO 94/29459, disclose the use of stress proteins and analogs for producing or enhancing an immune response or for inducing immune tolerance, for prophylaxis or therapy of autoimmune diseases and for treating or preventing infectious or cancers. A fusion protein is claimed comprising a stress protein fused to a protein against which an immune response is desired.
WO 95/25744 discloses microbial stress protein fragments containing epitopes homologous to related mammalian epitopes—used to treat and prevent inflammatory autoimmune diseases and to prevent transplant rejection. The protective epitopes are located in short peptides comprising 5-15 amino acid sequences regions of stress proteins, that are highly conserved between microorganisms and animals.
WO 97/11966 and WO 96/10039 disclose polypeptides of up to 21 amino acids, derived from microbial heat shock protein which are useful for prophylaxis or treatment of autoimmune diseases especially arthritis.
WO 96/16083 discloses a peptide 25 amino acids long, derived from the 10 kD heat shock protein (hsp10) of Mycobacterium tuberculosis which is useful in pharmaceutical products for the treatment of inflammatory pathologies, especially rheumatoid arthritis.
WO 91/02542 discloses the use of antigenic and/or immuno-regulatory material derived from mycobacterium vaccae and specifically hsp60, for treating chronic inflammatory disorders caused or accompanied by an abnormally high release of IL-6 and/or TNFα.
WO 96/18646 discloses peptides of 9-20 amino acids derived from Mycobacterial hsp60 used for treatment or prevention of autoimmune CNS diseases, e.g. multiple sclerosis, chronic inflammatory CNS disease and primary brain tumors.
WO 94/02509 discloses peptides of 7-30 amino acids derived from DR3-restricted epitope of Mycobacterial hsp60 used for treatment of HLA-DR3 related autoimmune diseases.
WO 00/27870 discloses peptides derived from Mycobacterial and rat hsp60 and vaccines comprising such peptides for immunization against autoimmune and inflammatory diseases.
U.S. Pat. No. 5,958,416 describes heats shock protein peptides and methods for modulating autoimmune central nervous system diseases.
WO 01/43691 discloses fragments and antagonists of Hsp60, capable of reducing or prevention the induction of a pro-inflammatory immune response of cells of the innate immune system by hsp60, for treatment of inflammatory and autoimmune diseases. The compounds disclosed inhibit the binding of hsp60 to the toll-like-receptor, and therefore reduce or prevent the induction of a consequent pro-inflammatory response.
Additional heat shock proteins, other than hsp60, have been disclosed as useful for treatment. For example, U.S. Pat. No. 5,348,945 discloses a method for reducing mortality in stressed tissue with heat shock protein for treatment of atherosclerosis, arterial restenosis and anoxic nerve damage using exogenous hsp70. Other inventions (e.g. JP 10212230, JP 09241159) disclose synthetic and natural compounds and extracts which inhibit the expression of proteins belonging to the hsp60 or hsp27 families and are therefore useful for treating autoimmune diseases and cancers.
Srivastava and colleagues have disclosed use of non-covalent complexes of hsp70, hsp90 or hsp96 together with an antigen for preventing and treating cancer and infectious diseases and for treatment of autoimmune diseases such as diabetes and multiple sclerosis. U.S. Pat. No. 6,322,790 discloses compositions and methods for eliciting an immune response using heat shock protein-peptide complexes in combination with adoptive immunotherapy, for prevention and treatment of neoplastic diseases and infectious diseases. In these methods the complex consists of a heat shock protein non-covalently bound to an antigenic molecule in combination with administering antigen presenting cells sensitized with complexes of hsps non-covalently bound to an antigenic molecule.
Toll Proteins and hsp60
It was recently discovered that hsp60 is a putative endogenous activator of Toll-like receptors in mammals (Ohashi et al. J. Immunol. 164, 558-61, 2000), while the previously described ligands for Toll-like receptors in mammalian cells are of microbial origin, which is in line with a function of these receptors in innate immune responses. This funding suggests that Toll-like receptors may not only have a function in innate immune defense against microbial pathogens but also serve physiological functions by interacting with endogenous ligands.
It is noteworthy that both Toll-like receptors and hsp60 are found early in phylogeny and both are of remarkably conserved structure. This suggested that their interaction is relevant and may also occur in more primitive organisms. Mammalian hsp60 usually is sequestered to the cell interior, in accordance with its ability to function as a chaperone. However, hsp60 becomes accessible when it is set free during necrosis of tissue during inflammation or when hsp60 is partially translocated to the plasma membrane in response to diverse types of stress. It was therefore proposed that autologous hsp60 may serve as a danger signal antigen to the innate immune system (Chen et al. ibid).
Cells of the Adaptive Immune System
The adaptive immune response plays a critical role in the eradication of pathogens. However, inappropriate responses to infection can cause severe pathology. The cells of the adaptive immune system are normally present as circulating cells in the blood and lymph, in an anatomically defined collection of lymphoid organs, and as scattered cells in virtually all tissues. The ability of the adaptive immune system to optimally perform its protective function is dependent on several properties of its constituent cells and tissues. The structure and function of the immune system is reviewed by Abbas et al. Cellular and Molecular immunology, Fourth Edition 2000, W. B. Saunders Company, Philadelphia.
Dendritic Cells and their Role in Immunity
Dendritic cells (DCs) are a family of bone marrow derived antigen-presenting cells (APCs) with an exquisite capacity to interact with T cells and modulate their responses. DCs patrol most non-lymphoid organs including epithelia (e.g. the skin and the mucosa of the intestines where DCs are called Langerhans cells (LCs)), the dermis and the interstitia of vascularized organs such as the heart and kidneys. DCs are also found in the blood and lymph and are present in all lymphoid organs. In the T cell areas of the lymphoid organs they are called interdigitating DCs. DCs from non-lymphoid tissues can migrate to secondary lymphoid organs, via the blood or lymph, bringing antigens to naive T cells from peripheral sites to which the latter cells are excluded. As a very generalized rule, if the DC presenting the antigen to the naive T cell is non-activated, the T cell will be tolerogenic. If the DC presenting the antigen to the naive T cell is activated then the T cell will be stimulated to produce a response. In other words, the first critical decision regulating the response of the immune system depends on whether or by what the dendritic cell has been activated.
The involvement of the heat shock proteins in modulation of immune responses is now well established, however, nowhere in the background art is it taught or suggested that exposure of dendritic cells to Hsp proteins alone (in the absence of antigen) or to isolated Hsp derived peptides can directly regulate the immune response, and that peptides and analogs of heat shock proteins may act directly at the dendritic cell level thereby leading to discrimination between Th1 and Th2 subsets of T cells.